Detection and localization of ApxI, -II and -III genes of Actinobacillus pleuropneumoniae in natural porcine pleuropneumonia in natural porcine pleuropneumonia by in situ hybridization

In situ hybridization techniques that employed a nonradioactive digoxigenin-labeled probe were used to detect and localize ApxI, II and III genes in tissue sections of pneumonic lung naturally infected with Actinobacillus pleuropneumoniae. In pigs infected with either serotype 2 or 6, a hybridization signal for apxIICA, apxIIICA, apxIBD, and apxIIIBD was detected, and in pigs infected with serotype 5, a hybridization signal for apxICA, apxIICA, and apxIBD was detected in the pneumonic lesions. A hybridization signal for apxIICA and apxIBD was detected in pigs infected with serotype 7. A strong hybridization signal for apx genes was seen in streaming degenerate alveolar leukocytes bordering zones of coagulative necrosis. Simultaneous detection of hybridization signals for the apxCA and apxBD genes provided scientific evidence that the expression of the apx genes could be potential indicators of the production of corresponding Apx toxins. This study demonstrates the expression of ApxI, II, and III genes in pneumonic lesions caused by A. pleuropneumoniae.     

Detection and localization of Mycoplasma hyopneumoniae DNA in lungs from naturally infected pigs by in situ hybridization using a digoxigenin-labeled probe.

Mycoplasma hyopneumoniae DNA was detected in 20 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled DNA probe. A 520-base-pair DNA probe targeting a reiterative sequence of the M. hyopneumoniae genome was generated by the polymerase chain reaction. All 20 pigs infected with M. hyopneumoniae had distinct and positive hybridization signals without background staining. A strong hybridization signal was detected mainly in the luminal surface of bronchial and bronchiolar lining epithelial cells, whereas no hybridization signal was seen in the cytoplasm of bronchial and bronchiolar lining epithelial cells. When hybridization signal was detected in the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole had peribronchiolar lymphoid hyperplastic tissues. Hybridization signals were not seen in the peribronchiolar lymphoid hyperplastic tissues. A less intense signal was detected in the interstitial and alveolar macrophages randomly scattered in the thickened alveolar septa and spaces. Hybridization signal was rarely detected in the type I pneumocytes. The in situ hybridization technique developed in this study was useful for detection of M. hyopneumoniae nucleic acids in tissues taken from naturally infected piglets and may be a valuable technique for studying the pathogenesis of M. hyopneumoniae infection.     

Expression of cyclooxygenase-2 in swine naturally infected with Actinobacillus pleuropneumoniae

Cyclooxygenase-2 (COX-2) was detected and localized in 15 pigs with naturally occurring pleuropneumonia using a 437-base pair digoxigenin-labeled cDNA probe in an in situ hybridization protocol. Histopathologic changes in the acute stage were characterized by coagulative necrosis of lung parenchyma, hemorrhage, vascular thrombosis, edema, fibrin deposition, and infiltration of lung parenchyma by neutrophils and alveolar macrophages in nine pigs. In chronic lesions, a thick layer of granulation tissue surrounded foci of pulmonary necrosis in six pigs. All 15 pigs infected with Actinobacillus pleuropneumoniae, confirmed by bacterial isolation, had distinct positive hybridization signals for COX-2 in bronchial, bronchiolar epithelial cells, alveolar macrophages, neutrophils, and type I pneumocytes. COX-2 expression was detected primarily in neutrophils from pigs with acute lesions and primarily in alveolar macrophages from pigs with chronic lesions. The results suggest that a prostanoid product of COX-2 is an important component of the inflammatory response to acute and chronic A. pleuropneumoniae infection.     

Actinobacillus pleuropneumoniae osteomyelitis in pigs demonstrated by fluorescent in situ hybridization.

Necrotizing osteomyelitis and fibrinopurulent arthritis with isolation of Actinobacillus pleuropneumoniae serotype 2 is reported in two pigs from a herd with lameness and mild coughing problems among 8 to 12-week-old pigs. Application of fluorescent in situ hybridization targeting 16S ribosomal RNA of A. pleuropneumoniae in formalin-fixed tissue was performed to verify the association of A. pleuropneumoniae with the bone and joint lesions. By in situ hybridization A. pleuropneumoniae was demonstrated as multiple microcolonies or single cells dispersed in focal fibrinonecrotizing pleuropneumonia, in joints with arthritis, and in bone necroses including lysis of growth plate and suppurative inflammation in the adjacent trabecular metaphysis, thus demonstrating that well-known infections manifest new, unusual lesions.     

Development of polymerase chain reaction and comparison with in situ hybridization for the detection of Haemophilus parasuis in formalin-fixed, paraffin-embedded tissues

DNA extraction and nested polymerase chain reaction (PCR) were developed for the detection of Haemophilus parasuis from formalin-fixed, paraffin-embedded tissues. The results for nested PCR were compared with those determined by in situ hybridization. The optimal results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested PCR is a reliable detection method. A distinct positive signal was detected in 20 pigs naturally infected with H. parasuis by in situ hybridization. The rate of agreement between nested PCR and in situ hybridization for the detection of H. parasuis in formalin-fixed, paraffin-embedded tissues was 100%. The nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection of H. parasuis with bacterial isolation.     

Localization of classical swine fever virus from chronically infected pigs by in situ hybridization and immunohistochemistry

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.     

A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions

The study describes a polymerase chain reaction (PCR) assay for the detection of Actinobacillus pleuropneumoniae. The test is based on the amplification of the omlA gene coding for an outer membrane protein of A. pleuropneumoniae. To test the specificity of the reaction, 19 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR assay. The detection threshold of the test was 10(2) A. pleuropneumoniae CFU/assay. The test was then applied to the detection of A. pleuropneumoniae from tonsillar biopsies and tracheobronchial lavage fluids of pigs without a culture step. The detection of A. pleuropneumoniae in these samples was performed by PCR, by conventional culture and by bacteriology with immunomagnetic beads. The number of samples that were found positive by PCR was almost three times higher than the number of samples from which A. pleuropneumoniae was isolated by both bacteriological techniques. The detection of A. pleuropneumoniae in these samples allowed us to demonstrate its aerosol transmission to pigs under experimental conditions. The trial involved 18 specific pathogen free pigs. Six pigs, infected with A. pleuropneumoniae, were located in a unit A, together with four non-infected animals (contact pigs). Eight non-infected pigs (reporter pigs) were located in a unit B, adjacent to A. We detected A. pleuropneumoniae in samples from infected animals but also from 'contact' (unit A) and 'reporter' (unit B) pigs. The results of this study show that the simple preparation of the samples followed by the PCR assay may be a useful tool for epidemiological studies.     

A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs.

Virus isolation, polymerase chain reaction (PCR), immunohistochemistry, and in situ hybridization were compared for the detection of porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) from experimentally and naturally coinfected pigs. All coinfected pigs developed postweaning multisystemic wasting syndrome (PMWS), characterized by sudden onset of depression and anorexia. Microscopically, granulomatous inflammation with intracytoplasmic inclusion bodies was present in lymph node from all coinfected pigs at 32 days postinoculation. Of the 200 tissues from 20 experimentally coinfected pigs evaluated, 99 and 58 tissues were positive for PCV2 and PPV, respectively, by 4 techniques. Virus isolation, PCR, immunohistochemistry, and in situ hybridization identified PCV2 infection in 137, 148, 103, and 129 tissues and PPV infection in 107, 132, 59, and 94 tissues. Of the 200 tissues from 20 naturally coinfected pigs evaluated, 109 and 45 tissues were positive for PCV2 and PPV, respectively, by 4 techniques. Virus isolation, PCR, immunohistochemistry, and in situ hybridization identified PCV2 infection in 144, 155, 113, and 139 tissues and PPV infection in 93, 109, 45, and 82 tissues. Because the characteristic microscopic lesions are important criteria for the diagnosis of clinical PMWS, immunohistochemistry and in situ hybridization for the detection of PCV2 and PPV in formalin-fixed, paraffin-embedded tissues provide confirmation of a histopathological diagnosis of PMWS.     

Utilization of a rate enhancement hybridization buffer system for rapid in situ hybridization for the detection of porcine circovirus in cell culture and in tissues of pigs with postweaning multisystemic wasting syndrome.

A rapid in situ hybridization (ISH) technique for the detection of porcine circovirus (PCV) nucleic acid in cell culture and formalin-fixed paraffin-embedded tissues was developed. A fluorescein-labeled RNA probe was transcribed from a plasmid containing 530 bp of the ORF1 of a PCV isolated from a pig with postweaning multisystemic wasting syndrome (PMWS). Hybridization using standard hybridization buffer was performed at 42 C for 16 hours and was compared to hybridization using rate enhancement hybridization (REH) buffer at 67 C for 2 hours. Hybridization was detected with an alkaline phosphatase-conjugated antifluorescein antibody. In both cultured cells and tissues from pigs with PMWS, the signal intensity and number of labeled cells in sections hybridized with REH buffer were equal to those of sections hybridized with standard hybridization buffer. The total time required for ISH using the REH buffer is 7-8 hours, thus making this protocol suitable for application in routine PCV diagnosis.     

In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods.

Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available.     

Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV).

A double in situ hybridization method for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV) genomes in the same tissue section was applied to lung tissues from 9 pigs in which PRRSV and PCV coinfection had been previously demonstrated. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-labeled antisense RNA probe for PCV, and hybridization was detected with anti-digoxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/diaminobenzidine, respectively. PRRSV and PCV genomes were identified in the same pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV-positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.     

Evaluation of Actinobacillus pleuropneumoniae diagnostic tests using samples derived from experimentally infected pigs

New serological tests have recently been introduced for Actinobacillus pleuropneumoniae diagnosis. No information is currently available on how these tests compare regarding the detection of antibodies from subclinically infected pigs. To answer this question, 80 pigs were randomly assigned to experimental groups infected with A. pleuropneumoniae serovars 1, 3, 5, 7, 10, 12, 15 and a non-inoculated control group. Blood samples and oropharyngeal swabs were collected prior to infection and for 7 consecutive weeks thereafter. Serum samples were tested using the Swinecheck(?) APP ELISA and the Multi-APP ELISA (University of Montreal). All pigs were euthanized at 49 days post-inoculation. Tonsil and lung samples were cultured for isolation and tested by PCR. The Multi-APP ELISA detected seroconversion 1 week earlier than the Swinecheck(?) APP ELISA with the earliest seroconversion detected at 1 week post-infection (serovar 10) and the latest at 3 weeks post-infection (serovar 1). Seroconversion at day 49 was serovar-dependent and varied from 4 (44%) positives detected in the serovar 10 group to 9 positives (100%) detected in the serovar 15 group. Thirty-one pigs were serologically positive for A. pleuropneumoniae at 49 days post-infection and only 15 still carried A. pleuropneumoniae on their tonsils based on PCR results. No cross-reactions were observed when serum samples were cross-tested using the Swinecheck(?) APP ELISA. A. pleuropneumoniae was successfully isolated from the lung of 2 pigs that developed pleuropneumonia, but was not isolated from tonsils due to heavy contamination by the resident flora. This study offers a comprehensive evaluation of the diagnostic tools currently available for detection of A. pleuropneumoniae subclinical infection.     

Multiplex nested PCR compared with in situ hybridization for the differentiation of porcine circoviruses and porcine parvovirus from pigs with postweaning multisystemic wasting syndrome

Multiplex nested polymerase chain reactions (PCRs) were developed for the simultaneous detection and differentiation of genomic material of porcine circovirus 1 (PCV1), porcine circovirus 2 (PCV2), and porcine parvovirus (PPV) in formalin-fixed, paraffin-embedded tissues. Multiplex conventional and nested PCR and in situ hybridization were compared for their ability to detect the 3 viruses in such tissues. Xylene deparaffinization followed by proteinase K digestion yielded DNA of sufficient quality for reliable and consistent PCR analyses. The DNA from PCV1, PCV2, and PPV was detected by both multiplex nested PCR and in situ hybridization in lymph-node tissue from 12 pigs experimentally co-infected with the 3 viruses, as well as in formalin-fixed, paraffin-embedded lymph-node tissue from 30 pigs with naturally occurring postweaning multisystemic wasting syndrome; the agreement rates for the 2 methods were 100% in both groups of pigs. Thus, multiplex nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for simultaneous detection of these 3 porcine viruses.     

A pathogenesis study of foot-and-mouth disease in cattle, using in situ hybridization.

Eight calves were exposed in an aerosol chamber to nebulized foot-and-mouth disease virus. Two control animals were exposed in a similar manner to cell culture media only. Animals were euthanized at intervals and various tissues examined by in situ hybridization using a biotinylated RNA probe corresponding to a portion of the viral gene coding for the polymerase enzyme. By this technique large amounts of viral nucleic acid were found in coronary band, interdigital cleft and tongue as early as six hours postexposure, indicating a very rapid delivery from the portal of entry to the predilection sites for lesion development. This occurred well before the onset of viremia which by virus isolation was not detectable until 30 hours postexposure. The in situ hybridization signal in these tissues decreased in intensity and extent with time to focally positive areas, occasionally surrounding a vesicle. Other epidermal sites not normally thought of as sites for foot-and-mouth lesion development, such as carpus and eyelid, also had some viral nucleic acid detectable at various time intervals. In the lung by in situ hybridization, alveolar septa had viral nucleic acid early in infection (6-18 h postexposure) while later (36-96 h postexposure), the in situ hybridization signal was prominent in alveolar macrophages.     

Chronologic localization of mycoplasma hyopneumoniae in experimentally infected pigs

The chronologic localization of Mycoplasma hyopneumoniae was examined by in situ hybridization in experimentally infected pigs for a period of 35 days after intratracheal inoculation. M. hyopneumoniae DNA was detected in bronchial and bronchiolar epithelial cells from infected pigs at 7, 14, 21, and 28 days postinoculation (DPI) and in alveolar and interstitial macrophages and type I pneumocytes from infected pigs at 14, 21, 28, and 35 DPI. Strong hybridization signals for M. hyopneumoniae were detected mainly at the luminal surface of bronchial and bronchiolar lining epithelial cells. When a hybridization signal was detected at the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole also exhibited peribronchiolar lymphoid cuffing. These observations suggested that the presence of M. hyopneumoniae in different tissues could be due to a difference in the duration of the infection.